Materials and Methods for the Treatment of Pathological Neovascularization in the Eye

ABSTRACT

The subject invention provides materials and methods useful in safely and effectively preventing pathological proliferation of blood vessels. The prevention of the over-proliferation of blood vessels according to the subject invention is particularly advantageous for treatment of certain ocular conditions including age-related macular degeneration (AMD), retinopathy of prematurity (ROP) and diabetic retinopathy. In preferred embodiments, the subject invention provides materials and methods for effective treatment of pathological ocular neovascularization using gene therapy. In a specific embodiment the materials and methods of the subject invention can be used to treat AMD.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 13/246,527, filed onSep. 27, 2011, and claims the priority benefit of U.S. ProvisionalApplication Ser. No. 61/386,889, filed Sep. 27, 2010, the contents ofeach of which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

The subject invention was made with government support under Grant No.R03 EY016119 (JB) awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

SEQUENCE LISTING

This application incorporates by reference in its entirety the SequenceListing entitled December2011_ST25.txt (1,880 bytes) which was createdDec. 8, 2011 and filed with the application on Jul. 18, 2014.

BACKGROUND OF INVENTION

People suffering from visual impairment face many challenges inperforming routine daily activities and/or may not be able to fullyenjoy the visual aspects of their surroundings. Of particular concernare visual impairments caused by damage to the retina, which occur inconditions such as age-related macular degeneration (AMD), diabeticretinopathy, and retinopathy of prematurity.

Age-related macular degeneration (AMD) is the leading cause of blindnessamong adults over the age of 60 in the Western world [Klein, Peto etal., (2004)] with approximately 2 million people suffering from AMD inthe US and over 7 million more people at risk (Friedman, O'Colmain etal., 2004). AMD is a multifactorial disease that affects the central (ormacular) portion of the retina, the light-sensitive tissue that linesthe back of the eye and is responsible for central vision and thedetection of fine detail (or acuity).

The acute loss of central vision associated with the wet, orneovascular, form of AMD is the result of unstable, new abnormal bloodvessels that grow into the back of the eye where they interfere withnormal vision. Prevention of this pathological process, termed choroidalneovascularization, or CNV, is the major target for treatment of the“wet” form of AMD. In AMD patients, CNV is strongly associated withhypoxia and chronic inflammation. These events typically precede CNV,and are thought to play an underlying role in the induction of theneovascular process.

There is a critical need for alternative and more effective treatmentsfor AMD because, for example, the existing treatment requires multiple,costly injections into the eye with the possibility of infection.

Diabetic retinopathy is a progressive disease characterized byabnormalities of the blood vessels of the retina, such as weakening ofthe blood vessel walls, leakage from the blood vessels, and bleeding andscarring around new vessels. Diabetic retinopathy results in impairmentof a person's vision causing severely blurred vision and, potentially,blindness. The World Health Organization indicates that diabetesafflicts 120 million people worldwide, and estimates that this numberwill increase to 300 million by the year 2025. Diabetic retinopathy is aform of visual impairment often suffered by diabetics.

Due to significant medical advancements, diabetics are able to live muchlonger than in the past. However, the longer a person has diabetes thegreater the chances of developing diabetic retinopathy. Affecting over5.3 million Americans, diabetic retinopathy is the leading cause ofblindness among adults in the United States. Annually, in the UnitedStates, between 12,000 and 24,000 people lose their sight because ofdiabetes.

While management of diabetic retinopathy has improved, risk ofcomplications, such as loss of visual acuity, loss of night vision andloss of peripheral vision, remains significant. Currently, laserphotocoagulation is the most effective form of therapy for advanceddisease. Unfortunately, current treatment options are inadequate and thedisease is often progressive even with successful glucose control.

Retinopathy of prematurity (ROP) is a disorder of retinal blood vesseldevelopment in the premature infant. Under normal development, bloodvessels grow from the back central part of the eye out toward the edges.In premature babies, this process is not complete and the abnormalgrowth of the vessels proliferates leading to scar tissue development,retinal detachment and possibly complete blindness.

ROP is the major cause of blindness in children under the age of seven.Improved care in the neonatal intensive care unit has reduced theincidence of retinopathy of prematurity in moderately premature infants.Ironically, however, increasing rates of survival of very prematureinfants, who would have had little chance of survival in the past, haveincreased the occurrence of ROP.

Current research shows promise that the prevention of retinal bloodvessel damage, which marks retinopathy, may be achieved by theutilization of certain compounds. For example, it has been demonstratedthat, in retinal epithelial cells, glutamine deprivation can lead toupregulation of vascular endothelial growth factor (VEGF) expression(Abcouwer S. et al., “Response of VEGF expression to amino aciddeprivation and inducers of endoplasmic reticulum stress,” InvestOphthalmol Vis Sci, August 2002, pp. 2791-8, Vol. 43, No. 8). Most sickpremature infants are deprived of glutamine during the time they receivesupplemental oxygen, a known predisposing factor in the development ofROP. The over expression of VEGF during this time period is also thoughtto be involved in the pathogenesis of ROP.

Endostatin, a 20 kDa proteolytic fragment of the carboxy terminus ofcollagen XVIII, was discovered in 1997. It was the first endogenousinhibitor of angiogenesis identified as a fragment of a matrix protein.By binding multiple receptors and initiating numerous intracellularpathways, endostatin elicits a broad spectrum of anti-proliferative,anti-migratory, and apoptotic effects on the endothelial cells that linethe walls of blood vessels.

Although the delivery of anti-angiogenic agents has been shown to resultin the destruction of newly formed blood vessels the constant presenceof an anti-angiogenic agent, such as endostatin, may result in long-termdeleterious effects on normal vessels in the eye.

BRIEF SUMMARY

The subject invention provides materials and methods useful in safelyand effectively preventing or treating pathological proliferation ofblood vessels in the eye. The prevention of the over-proliferation ofblood vessels according to the subject invention is particularlyadvantageous for treatment of certain ocular conditions includingage-related macular degeneration (AMD), retinopathy of prematurity (ROP)and diabetic retinopathy.

In preferred embodiments, the subject invention provides materials andmethods for effective treatment of pathological ocularneovascularization using gene therapy. In a specific embodiment thematerials and methods of the subject invention can be used to treat AMD.

Advantageously, the novel gene therapy strategies of the subjectinvention provide regulated production of an angiogenesis-inhibitingprotein, such as endostatin. Specifically, the use of ahypoxia-regulated, retinal pigment epithelial cell-specific vectorprovides a pathology-initiated regulation of the expression of anangiogenesis inhibitor. The administration of this vector and thesubsequent regulated expression of the inhibitor can be used to treatpathological ocular neovascularization, including, for example, the mostdevastating effects of “wet” AMD.

In accordance with the subject invention, the angiogenesis inhibitor isregulated by a promoter that turns on the gene therapy vector to producethe inhibitor only in pathologic areas of neovascularization. Thus, theproduction of the inhibitor is regulated, i.e., is not produced all ofthe time and at all places. The result is a more efficacious delivery ofthe inhibitor leading to an improved outcome compared to therapies wherea medicament is periodically injected into the eye or is constantlyreleased from a gene therapy vector.

An additional advantage of the cell-specific, regulated vector of thesubject invention is that the mechanism of action to reduceneovascularization has been established. This facilitates furtherrefinements to the technique to achieve even better results.

The compositions of the subject invention can be formulated according toknown methods for preparing pharmaceutically useful compositions. Ingeneral, the compositions of the subject invention will be formulatedsuch that an effective amount of the bioactive agent is combined with asuitable carrier in order to facilitate effective administration of thecomposition.

In accordance with the invention, pharmaceutical compositions comprisean active agent, and one or more non-toxic, pharmaceutically acceptablecarriers or diluents. Pharmaceutical carriers or excipients may containinert ingredients that do not interact with the active agent, oringredients that do interact with the active agent but not in a fashionso as to interfere with the desired effect. In general, the formulationsare prepared by uniformly and intimately bringing into association theactive ingredient with a liquid carrier.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a hypoxia responsive element (HRE) sequence usefulaccording to the subject invention.

SEQ ID NO:2 is a hypoxia responsive element (HRE) sequence usefulaccording to the subject invention.

SEQ ID NO:3 is a neuron restrictive silencer element (NRSE) sequenceuseful according to the subject invention.

SEQ ID NO:4 is a primer sequence useful according to the subjectinvention.

SEQ ID NO:5 is a primer sequence useful according to the subjectinvention.

SEQ ID NO:6 is a primer sequence useful according to the subjectinvention.

SEQ ID NO:7 is a primer sequence useful according to the subjectinvention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the vector design for regulated production ofendostatin in hypoxic retinal pigment epithelial (RPE)(scAAV2-HRSE-6×HRE-RPE65-Endostatin) and the constitutive endostatinvector construct (scAAV2-CMV-Endostatin). The regulated endostatinconstruct contains, in the 5′ to 3′ direction, a hypoxia-regulatedsilencing element (HRSE) containing the tandem alternating repeats of 3copies neuron restrictive silencer element (NRSE) with 3 copies of theHRE-HIF1 enhancer element, 6 tandem copies of the HRE-HIF1 enhancerelement, and the RPE promoter.

FIG. 2 shows rtPCR analysis of virus-derived hrEndostatin mRNAtranscripts from ARPE-19 cells transduced with scAAV-HRSE-6×HRE-RPE65and subjected to either 24 hours of hypoxia (1% O₂) or normoxia (21%O₂). RtPCR of mRNA from untransduced ARPE-19 cells, exposed to the samehypoxic and normoxic conditions, was used to ensure that endogenoustranscripts were not inadvertently amplified. A 682 bp band indicatespresence of viral transcripts. The results indicate minimal viralexpression in normoxia, along with a strong hypoxic-induction of viraltranscripts. Untransduced cells lacked the presence of virus-derivedendostatin transcripts in hypoxia.

FIG. 3 shows representative confocal micrographs of CNV lesions from RPEflatmounts. The top row shows CNV lesions of mice subretinally injectedwith vehicle control. The middle row shows CNV lesions of micesubretinally injected with scAAV2-HRES-HRE-RPE65-Endostatin (Reg Endo).The bottom row shows CNV lesions of mice subretinally injected withscAAV2-CMV-Endostatin. Arrows indicate CNV lesion boundary.Green=fluoroscein-conjugated tomato lectin perfused vasculature;blue=DAPI nuclear stain. Scalebars=100 um.

FIG. 4A shows that the mean CNV area in eyes treated with RegulatedEndostatin virus or CMV Endostatin was significantly lower than that ofvehicle-treated control eyes. (*P<0.001, t-test; n=# of lesionsmeasured). FIG. 4B shows the mean CNV area of theAAV2-HRSE-HRE-RPE65-Endostatin injected (white bars) and thecontralateral, vehicle injected eyes (black bars) from the same mouse.Only mice that had at least 2 countable lesions per eye are shown. FIG.4C compares mean CNV area in all eyes treated with regulated endostatinwith those from the contralateral, vehicle injected eyes (from B).(*P<0.001; paired T-test).

FIG. 5 shows human endostatin concentrations measured by ELISA. Mice aresubretinally injected with the AAV2-Reg-Endo vector, the AAV2-CMV-Endovector, or vehicle (VC). Each sample represents 4 homogenized posterioreyecups pooled for each of the 3 treatment groups at 3 time points:pre-laser (5 days post-injection), 3 days post-laser, and 2 weekspost-laser. The results show that there is a conditional silencing ofthe reg-endo vector before laser injury, then enhanced activationsubsequent to laser injury, and a return to baseline two weekspost-laser injury. Endostatin concentrations in mice treated with theCMV-endo vector are elevated prior to laser injury, and remain elevatedthrough the 2 week post-laser time point.

FIG. 6 shows CNV lesion histology light micrographs of CNV lesions ofthe mice 3 days post-laser injury. Images are derived from mice injectedwith the regulated-endostatin injected vector, 6 months post-injection(a & b), and contralateral vehicle control injected eyes (c & d) fromthe same mouse. Hematoxalin & Eosin; b & d are higher magnification of a& c. Scale bars=100 μm.

FIG. 7 shows immunohistochemical localization of human endostatin(green) in cryosections through CNV lesions in eyes injected withvehicle (a, b) or the regulated endostatin vector (c, d). The eyes wereharvested 3 days post-laser injury. b & d are higher magnificationimages of a & c. Arrows indicate RPE/Choroid interface. Asteriskindicates center of lesion. Scale bars=100 μm; ONL, outer nuclear layer;RPE, retinal pigment epithelium.

DETAILED DISCLOSURE

There is a critical need for new materials and methods for protectingnormal retinal tissue from cell damage due to abnormalneovascularization, as well as for retarding further growth of newvessels after neovascularization has already occurred. In oneembodiment, the subject invention provides a novel form of gene therapywherein an anti-angiogenic agent is produced only at the site ofabnormal new blood vessels and only under pathological conditions.

In a specific embodiment of the subject invention, gene therapy can beused for preventing the proliferation of abnormal retinal blood vesselsin a patient. Specifically, as described herein, the compounds producedby the vector of the subject invention are effective for inhibitingpathological vascular proliferation.

Accordingly, the subject invention is useful prophylactically andtherapeutically for treating animals, including humans and othermammals, at risk for pathological vascular proliferation includingvascular retinopathy and vasculature associated with tumors. Thesecompositions can be administered to the elderly, premature infants,diabetics, or others who are at risk for retinal disease.

In a preferred embodiment, the method of the subject invention utilizesa gene therapy vector that drives robust protein production in hypoxicretinal pigment epithelial (RPE) cells, but not in normoxic cells.Advantageously, the vector is cell- and tissue-specific since the vectoris only expressed in RPE cells.

Endostatin is specifically exemplified herein as an angiogenesisinhibitor. Other angiogenesis inhibitors currently known or that will bedeveloped in the future and can be used. Other known angiogenesisinhibitors include, for example, VEGF receptors, angiostatin, and PF-4.

The RPE-specific gene therapy vector of the subject invention has beenfound to significantly reduce neovascularization in a laser-inducedmurine model of choroidal neovascularization (an accepted animal modelof AMD). This method provides a unique treatment both prophylacticallyand after neovascularization is apparent in the early stages of AMD. Thegene therapy vector providing regulated production of endostatin can bedelivered to AMD patients during the early stages of the disease, whichmakes this approach relatively inexpensive, more effective and lessinvasive. In a specific embodiment, the vector can be used to treat themost devastating form of AMD, termed the “Wet” form because it isaccompanied by the growth of abnormal blood vessels into the subretinalspace, eventually leading to massive photoreceptor cell death in thecentral, macular region of the retina resulting in blindness.

Hypoxia-Regulated, Tissue-Specific Expression Vector

In one aspect, the subject invention provides an expression vector forprevention and/or treatment of ocular neovascularization as well asdiseases associated with ocular neovascularization. In one embodiment,the expression vector comprises a hypoxia-responsive element, anocular-specific promoter, and a transgene encoding a therapeuticmolecule, wherein the transgene is operably linked to theocular-specific promoter and the transgene is placed under the controlof the hypoxia-responsive element. The hypoxia-responsive elementupregulates gene expression under hypoxia but not under normoxia. Theocular-specific promoter selectively drives gene expression in thetarget ocular tissue associated with neovascularization.

Ocular tissue associated with neovascularization include tissues having,or at risk of developing, neovascularization. In one embodiment, thegene therapy is selectively delivered to hypoxic foci in tissues, whichfoci are predisposed to develop neovascularization. In one embodiment,target ocular tissues at risk of developing neovascularization aretissues under hypoxic conditions, ischemnia, and/or chronicinflammation, which usually precede neovascularization.

The term “hypoxia” or “hypoxic condition,” as used herein, refers to apathological condition in which the oxygen concentration in oculartissues is less than 10%, 8%, 5%, 3%, 1%, or 0.5% oxygen concentration;as a result, the ocular tissue is deprived of oxygen supply. Undernormoxic conditions, the oxygen concentration of ocular tissues is about20%.

In one embodiment, the hypoxia-responsive element is a Hypoxia ResponseElement (HRE). HRE binds to a transcription factor—hypoxia-induciblefactor-1 (HIF-1), which is a basic helix-loop-helix protein formed byheterodimerization of HIF-1α and HIF-1β. Under normoxic conditions,HIF-1α is rapidly degraded by the proteosome, whereas HIF-1β isunregulated. Under hypoxic conditions, heterodimerization of HIF-1α andHIF-1β occurs; HIF1 heterodimer translocates into the nucleus to bindHREs and upregulate gene expression. Use of a HRE is highly advantageousfor selective expression of a transgene in cells and tissues underconditions of hypoxia.

In one embodiment, the hypoxia-responsive element comprises the coreconsensus sequence for the HRE—(A/G)CGT(G/C)C (SEQ ID NO:1), whichoccurs in the 5′ or 3′ flanking sequences of greater than 60hypoxia-regulated genes identified to date. The inclusion of HREconsensus sequences provides an endogenous, exquisitely sensitiveregulatory pathway as a physiologic switch for hypoxia-regulateddelivery of a therapeutic gene. In one embodiment, thehypoxia-responsive element comprises SEQ ID NO:2.

In one embodiment, the expression vector comprises multiple (such as 2,3, 4, 5, 6, 7, 8, 9, or more) copies of the hypoxia-responsive element.The hypoxia-responsive element can be placed upstream or downstream ofthe promoter sequence. In one embodiment, one or multiple copies ofhypoxia-responsive element are placed upstream of the promoter sequence.

In a further embodiment, the expression vector further comprises anaerobic silencer—a regulatory element that silences gene expressionunder normoxic conditions. As a result, the expression of the transgeneis conditionally silenced under aerobic conditions, while is robustlyinduced under hypoxic conditions. In a specific embodiment, the aerobicsilencer is derived from neuron-restrictive silencer element (NRSE).

In one embodiment, the expression vector comprises a hypoxia-regulatedsilenced element (HRSE) comprising one or more copies (such as 2, 3, 4,5, 6, 7, 8, 9, or more copies) of the hypoxia-responsive element and/orthe aerobic silencer. In another embodiment, copies of thehypoxia-responsive element and the aerobic silencer are placed inalternating tandem order.

The aerobic silencer can be placed upstream or downstream of thepromoter sequence, or upstream or downstream of the hypoxia-responsiveelement. In one embodiment, the aerobic silencer is placed upstream ofthe promoter sequence and the hypoxia-responsive element. In anotherembodiment, the aerobic silencer is placed downstream of the promotersequence and the hypoxia-responsive element.

In another embodiment, the aerobic silencer is placed upstream of thepromoter sequence but downstream of the hypoxia-responsive element. Inanother embodiment, the hypoxia-responsive element is placed upstream ofthe promoter sequence but downstream of the aerobic silencer.

The ocular-specific promoter can be derived from the promoter region ofa gene that is only, or selectively, expressed in the ocular tissue ofinterest (e.g., at or near the site of pathological neovascularization,under hypoxic conditions, and/or inflammation). The ocular tissues ofinterest include, for example, retinal cells, retinal epithelial cells,Muller cells, choroid cells, etc.

The term “tissue-specific promoter,” as used herein, refers to apromoter sequence that only, or selectively, drives gene expression inthe specific tissue type where the gene expression is desired, whereasthe promoter sequence does not drive gene expression, or drivesexpression to a much lesser extent, in other types of tissues. Incertain embodiments, the promoter is specific for a certain type of theocular tissue, such as retinal cells, retinal epithelial cells, Mullercells, choroid cells, etc. For example, a RPE-specific promoter onlydrives gene expression in RPE cells, and leaving other types of eyetissues unmodified by transgene expression.

In certain embodiments, the ocular-specific promoter can be derived fromthe promoter sequence of RPE-65, which is selectively expressed inretinal pigment epithelium (RPE) cells; the vitelliform maculardystrophy (VMD2) promoter that is specific for RPE-specific expression;the promoter sequence of BFSP1—an eye lens-specific gene; the promotersequence of glial fibrilary acidic protein gene (GFAP), which isselectively expressed in Muller cells and retinal glial cells. Inanother embodiment, the ocular-specific promoter is not the GFAPpromoter.

Preferably, the hypoxia-responsive element, the aerobic silencer, andthe promoter sequence are of mammalian origin, or more preferably, ofhuman origin. In an alternative embodiment, the promoter, itself,comprises a hypoxia-responsive element and/or aerobic silencer. In anembodiment, the promoter sequence is placed upstream of the transgene.

In certain embodiments, the therapeutic molecule is an angiogenesisinhibitor (also referred to as anti-angiogenic agent). As used herein,the term “angiogenesis” refers to the generation of new blood vesselsinto a tissue or organ. Under normal physiological conditions, humans oranimals undergo angiogenesis only in very specific restrictedsituations. For example, angiogenesis is normally observed in woundhealing, fetal and embryonal development, and formation of the corpusluteum, endometrium and placenta. The term “anti-angiogenic activity”refers to the capability of an agent or composition to inhibit theformation of new blood vessels.

Angiogenesis inhibitors useful according to the subject inventioninclude, but are not limited to, endostatin, fibroblast grow factor(FGF) or VEGF receptors, angiostatin, pigment epithelium-derived factor(PEDF), platelet factor 4 (PF-4), and the amino terminal fragment (ATF)of urokinase containing an elongation factor G (EFG)-like domain. In oneembodiment, angiostatin is derived from the amino-terminal fragment ofplasinogen, and comprises the anti-angiogenic fragment of angiostatinhaving kringles 1 to 3. Endostatin is specifically exemplified herein asan angiogenesis inhibitor. In preferred embodiments, the angiogenesisinhibitors, such as endostatin, are of human origin.

Genes encoding other anti-angiogenesis protein can also be used. Suchgenes include, but are not limited to, genes encoding inhibitors of FGFor VEGF, stroam1 derived factor 1 (SDF-1), and metalloproteinaseinhibitors such as BB94.

The nucleic acid constructs can be incorporated into vectors suitablefor gene therapy. The expression vector of the subject invention can bea plasmid, or preferably a suitable viral vector. Viral vectors usefulfor performing the subject invention include recombinantadeno-associated virus (rAAV)-based vectors; adenoviral (Ad) vectors;AAV-adenoviral chimeric vectors; retroviral vectors such as lentiviralvectors, human T-cell lymphotrophic viral vectors; and herpes simplexvirus (HSV)-based viral vectors. In certain specific embodiments, theexpression vector is an adenovirus-based vector, a recombinantadeno-associated virus (rAAV)-based vector, or a lentiviral vector.

Preferred embodiments of the vectors are recombinant adeno-associatedvirus (rAAV) vectors. In certain specific embodiment, the AAV vectorsare selected from AAV1, AAV2, AAV3, AAV5, AAV5, AAV6, or AAV7. In someembodiments optimized for efficient transduction of retinal cells andrapid onset of expression, the strain rAAV 2/1 is preferred.

In a specific embodiment, the subject invention provides an AAV vectorcomprising an endostatin gene, wherein the endostatin gene is operablylinked to a RPE65 promoter, and the endostatin gene is placed under thecontrol of HRSE and HRE.

Gene Therapy for Ocular Neovascularization

In another aspect, the subject invention provides a method of preventionand/or treatment of ocular neovascularization as well as diseasesassociated with ocular neovascularization, via the delivery of theexpression vector of the subject invention using gene therapy. In oneembodiment, the method comprises administering, into a target oculartissue of the subject, an expression vector comprising a transgeneencoding a therapeutic molecule, wherein the transgene is operablylinked to an ocular-specific promoter and is placed under the control ofa hypoxia-responsive element, wherein the ocular-specific promoterselectively drives gene expression in the ocular tissue associated withneovascularization, and the hypoxia-responsive element upregulates geneexpression under hypoxia but not under nomoxia.

In one embodiment, the expression vector, or a therapeutic compositioncomprising the expression vector is delivered into, or near, the area ofneovascularization, where the pathological proliferation of new bloodvessels occur. In another embodiment, the expression vector, or atherapeutic composition comprising the expression vector can also bedelivered to a site that is at risk for developing neovascularization.

The term “subject” or “patient,” as used herein, describes an organism,including mammals such as primates, to which treatment with thecompositions according to the subject invention can be provided.Mammalian species that can benefit from the disclosed methods oftreatment include, but are not limited to, apes, chimpanzees,orangutans, humans, monkeys; domesticated animals such as dogs, cats,horses, cattle, pigs, sheep, goats, and chickens; and other animals suchas mice, rats, guinea pigs, and hamsters.

In one embodiment, the subject or patient has pathological ocularneovascularization such as chorodial neovascularization, which can bedetected using angiography, e.g., fluorescein angiography, alone or incombination with indocyanine-green angiography.

The term “treatment” or any grammatical variation thereof (e.g., treat,treating, and treatment etc.), as used herein, includes but is notlimited to, ameliorating or alleviating a symptom of ocularneovascularization, reducing, suppressing, inhibiting, lessening, oraffecting the progression, severity, and/or scope of ocularneovascularization.

The term “prevention” or any grammatical variation thereof (e.g.,prevent, preventing, and prevention etc.), as used herein, includes butis not limited to, delaying the onset of ocular neovascularization.Prevention, as used herein, does not require the complete absence ofsymptoms.

As demonstrated herein, the subject invention significantly arrests orretards ocular neoveascularization. Pathological ocularneovascularization that can be prevented and/or treated in accordancewith the subject invention can be from any region of the eye. In certainpreferred embodiments, the subject invention is used to prevent and/ortreat choroidal neovascularization as well as diseases and conditionsassociated with choroidal neovascularization. Diseases and conditionsassociated with choroidal neovascularization include, for example,age-related macular degeneration, histoplasmosis, myopic degeneration,choroidal rupture, photocoagulation, choroidal hemangioma, choroidalnonperfusion, choroidal osteomas, choroideremia, retinal detachment,neovascularization at ora serrata, punctate inner choroidopathy,radiation retinopathy, and retinal cryoinjury.

In addition, the subject invention can also be used to prevent and/ortreat corneal neovascularization as well as diseases and conditionsassociated with corneal neovascularization, such as corneal dystrophies.In addition, the subject invention can be used to prevent and/or treatretinal neovascularization as well as diseases and conditions associatedwith retinal neovascularization including, for example, diabeticretinopathy and retinopathy of prematurity.

Construction of Vectors and Expression Constructs

Vectors useful according to the subject invention can be constructed asdescribed, for example, by Doughetry et al. (Christopher J. Dougherty,George W. Smith, C. Kathleen Dorey, Howard M. Prentice, Keith A.Webster, Janet C. Blanks, “Robust hypoxia-selective regulation of aretinal pigment epithelium-specific adeno-associated virus vector”Molecular Vision 14:471-480 (Mar. 7, 2008), which is incorporatedherein, in its entirety, by reference.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid. Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors.” In general, expressionvectors of utility in recombinant DNA techniques are often in the formof “plasmids,” which refer to circular double stranded DNA loops which,in their vector form are not bound to the chromosome.

Vectors may also be viral vectors wherein the viral vector is selectedfrom the group consisting of a lentivirus, adenovirus, adeno-associatedvirus and virus-like vectors. The vector may also be a lipid vesicle orliposome wherein the DNA is surrounded by a lipid emulsion that is takenup by the cell. The invention is intended to include such other forms ofexpression vectors which serve equivalent functions and which becomeknown in the art subsequently hereto. An “expression vector” is a vectorcapable of expressing a DNA (or cDNA) or RNA molecules cloned into thevector and, in certain cases, producing a polypeptide or protein.Appropriate transcriptional and/or translational control sequences areincluded in the vector to allow it to be expressed in a cell.

As used herein, the term “operably linked” refers to a juxtaposition ofthe components described wherein the components are in a relationshipthat permits them to function in their intended manner. In general,operably linked components are in contiguous relation.

Expression constructs of the invention may optionally contain atranscription termination sequence, a translation termination sequence,signal peptide sequence, and/or regulatory elements. Transcriptiontermination regions can typically be obtained from the 3′ untranslatedregion of a eukaryotic or viral gene sequence. Transcription terminationsequences can be positioned downstream of a coding sequence to providefor efficient termination. Signal peptides are a group of short aminoterminal sequences that encode information responsible for therelocation of an operably linked peptide to a wide range ofpost-translational cellular destinations, ranging from a specificorganelle compartment to sites of protein action and the extracellularenvironment. Targeting a peptide to an intended cellular and/orextracellular destination through the use of operably linked signalpeptide sequence is contemplated for use with the peptides of theinvention. Unique restriction enzyme sites can be included at the 5′ and3′ ends of the expression construct to allow for insertion into apolynucleotide vector.

Expression of the cloned sequences occurs when the expression vector isintroduced into an appropriate host cell. If a eukaryotic expressionvector is employed, then the appropriate host cell would be anyeukaryotic cell capable of expressing the cloned sequences. Vectorsinclude chemical conjugates such as described in WO 93/04701, which hasa targeting moiety (e.g. a ligand to a cellular surface receptor), and anucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNAor RNA viral vector), fusion proteins such as described inPCT/US95/02140 (WO 95/22618) which is a fusion protein containing atarget moiety (e.g. an antibody specific for a target cell) and anucleic acid binding moiety (e.g. a protamine), plasmids, phage etc. Thevectors can be chromosomal, non-chromosomal or synthetic.

The vectors can be introduced by standard techniques, e.g., infection,transfection, transduction or transformation. Examples of modes of genetransfer include for example, naked DNA calcium phosphate precipitation,DEAE dextran, electroporation, protoplast fusion, lipofection, cellmicroinjection and viral vectors.

“Regulatory region” means a nucleic acid sequence which regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin (responsible for expressing different proteins oreven synthetic proteins). Regulatory regions include origins ofreplication, RNA splice sites, enhancers, transcriptional terminationsequences, signal sequences which direct the polypeptide into thesecretory pathways of the target cell, and promoters.

A regulatory region from a “heterologous source” is a regulatory regionthat is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are constructed by one having ordinary skillin the art.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and in the case of coding sequences, thecassette and sites of insertion are chosen to ensure insertion of thecoding sequences in the proper reading frame for transcription andtranslation.

Heterologous DNA refers to DNA not naturally located in the cell, or ina chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A “nucleic acid” is a polymeric compound comprised of covalently linkedsubunits called nucleotides. Nucleic acid includes polyribonucleic acid(RNA) and polydeoxyribonucleic acid (DNA), both of which may besingle-stranded or double-stranded. DNA includes cDNA, genomic DNA,synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides ornucleic acid sequence that encodes a protein is called the sensesequence.

A DNA “coding sequence” is a DNA sequence that is transcribed andtranslated into a polypeptide in a cell in vitro or in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus.Alternative 5′ terminae are also possible for certain genes.Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. Within the promoter sequence will be found atranscription initiation site (conveniently defined for example, bymapping with nuclease SI), as well as protein binding domains (consensussequences) responsible for the binding of RNA polymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then optionallytrans-RNA spliced and translated into the protein encoded by the codingsequence.

As used herein, the term “downstream,” when used in reference to adirection along a nucleotide sequence, means in the direction from the5′ to the 3′ end. Similarly, the term “upstream” means in the directionfrom the 3′ to the 5′ end.

As used herein, the term “gene” means the gene and all currently knownvariants thereof and any further variants which may be elucidated.

The therapeutic genes are incorporated into expression vectors,preferably, viral vectors. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. Defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Thus, a specifictissue can be specifically targeted. Examples of particular vectorsinclude, but are not limited to, a defective herpes virus 1 (HSV1)vector [Kaplitt et al., Molec. Cell. Neurosci. 2:320-330 (1991)],defective herpes virus vector lacking a glyco-protein L gene [PatentPublication RD 371005 A], or other defective herpes virus vectors[International Patent Publication No. WO 94/21807; International PatentPublication No. WO 92/05263; an attenuated adenovirus vector, such asthe vector described by Stratford-Perricaudet et al. [J. Clin. Invest.90:626-630 (1992); see also La Salle et al., Science 259:988-990(1993)]; and a defective adeno-associated virus vector [Samulski et al.,J. Virol. 61:3096-3101 (1987); Samulski et al., J. Virol. 63:3822-3828(1989); Lebkowski et al., Mol. Cell. Biol. 8:3988-3996 (1988)].

Adeno-associated viruses (AAV) are small, single-stranded DNA viruses(K. I. Berns, Parvoviridae: the viruses and their replication, p.1007-1041, in F. N. Fields et al., Fundamental Virology, 3rd ed., vol.2, (Lippencott-Raven Publishers, Philadelphia, Pa.) (1995)). Mostserotypes of AAV vectors can be used for gene therapy in the eye (e.g.,see Aurricchio et al. 2001 and Yang et al. 2002). Specifically, thereare at least eight AAV serotypes with varying degrees of gene transferefficiencies in vivo and varying unset of efficiencies. The entiresequences of AAV1, 2, 3, 4, 5, 6 have been determined, and thehomologies of various serotype genomes are between 52 and 82%(Bantel-Schaal U., and H. zur Hausen. J. Virol. 1999, 73: 939-947,Parvoviridae, Intervirology 5: 83-92)(Bantel-Schaal U., and H. zurHausen. 1998. Virology 134: 52-63) (Rutledge. EA., CL. Halbert, and DW.Russell. 1998. J. Virol. 72: 309-319). In addition, new AAV serotypes,such as AAV7 and AAV8, etc, have been reported.

The use of vectors derived from the AAVs for transferring genes in vitroand in vivo has been described (see WO 91/18088; WO 93/09239; U.S. Pat.No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488 528). These publicationsdescribe various AAV-derived constructs in which the rep and/or capgenes are deleted and replaced by a gene of interest, and the use ofthese constructs for transferring the said gene of interest in vitro(into cultured cells) or in vivo, (directly into an organism). Thereplication defective recombinant AAVs according to the invention can beprepared by cotransfecting a plasmid containing the nucleic acidsequence of interest flanked by two AAV inverted terminal repeat (ITR)regions, and a plasmid carrying the AAV encapsidation genes (rep and capgenes), into a cell line which is infected with a human helper virus(for example an adenovirus). The AAV recombinants which are produced arethen purified by standard techniques.

The invention contemplates the use of an AAV-derived recombinant viruswhose genome encompasses a sequence encoding a nucleic acid encoding atherapeutic transgene (such as an angiogenesis inhibitor) flanked by theAAV ITRs. The invention also relates to a plasmid encompassing asequence encoding a nucleic acid encoding an anti-angiogenic factorflanked by two ITRs from an AAV. Such a plasmid can be used as it is fortransferring the nucleic acid sequence, with the plasmid, whereappropriate, being incorporated into a liposomal vector (pseudo-virus).

The general procedures for use of adeno-associated virus (AAV) as avector for gene therapy are known in the art, such as described in U.S.Pat. Nos. 7,037,713; 6,953,575; 6,897,063; 6,764,845; 6,759,050;6,710,036; 6,610,290; 6,593,123; 6,582,692; 6,531,456; 6,416,992;6,207,457; and 6,156,303.

The construction of an AAV vector can be performed using methods knownin the art, such as described in Flotte T R. Adeno-associatedvirus-based gene therapy for inherited disorders. Pediatr Res. 2005December; 58(6):1143-7; Goncalves M A. Adeno-associated virus: fromdefective virus to effective vector, Virol J. 2005 May 6; 2:43; Surace EM, Auricchio A. Adeno-associated viral vectors for retinal genetransfer. Prog Retin Eye Res. 2003 November; 22(6):705-19; Mandel R J,Manfredsson F P, Foust K D, Rising A, Reimsnider S, Nash K, Burger C.Recombinant adeno-associated viral vectors as therapeutic agents totreat neurological disorders. Mol. Ther. 2006 March; 13(3):463-83.).

In addition, a reporter gene can be inserted into the vector for thedetection of transgene expression. A reporter gene can be detectable byany number of techniques, including by fluorescence detection,calorimetric detection or immunologic detection. Particularly preferredmarker genes encode green fluorescent protein and variants thereof.

Purification of recombinant AAV (rAAV) vectors can be accomplished usingtechniques known in the art, such as heparin sulfate-based columns,which can substantially eliminate adenovirus contamination (Clark etal., 1999; Zolotukhin et al., 1999); U.S. Pat. Nos. 6,143,548 and6,146,874). Recombinant AAV can also be purified double CsCl bandingmethod (Rolling & Samulski, 1995).

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the subject invention, to usingtype 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses ofanimal origin (see WO94/26914). Those adenoviruses of animal originwhich can be used within the scope of the subject invention includeadenoviruses of canine, bovine, murine (example: Mavl, Beard 75 (1990)81), ovine, porcine, avian, and simian (example: SAV) origin. Theadenovirus of animal origin is a canine adenovirus, more preferably aCAV2 adenovirus (e.g., Manhattan or A26/61 strain (ATCC VR-800), forexample).

Preferably, the replication defective adenoviral vectors of theinvention comprise the ITRs, an encapsidation sequence and the nucleicacid of interest. Still more preferably, at least the E1 region of theadenoviral vector is non-functional. The deletion in the E I regionpreferably extends from nucleotides 455 to 3329 in the sequence of theAd5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3Afragment). Other regions may also be modified, in particular the E3region (WO95/02697), the E2 region (WO94/28938), the E4 region(WO94/28152, WO94/12649 and WO95/02697), or in any of the late genesL1-L5.

The replication defective recombinant adenoviruses according to theinvention can be prepared by any technique known to the person skilledin the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham,EMBO J. 3 (1984) 2917). In particular, they can be prepared byhomologous recombination between an adenovirus or modified adenovirusgenome and a plasmid which carries, inter alia, the DNA sequence ofinterest. Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques, which are well known to one ofordinary skill in the art.

In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in U.S. Pat. No. 5,399,346; Mann et al.,1983, Cell 33:153; U.S. Pat. No. 4,650,764; U.S. Pat. No. 4,980,289;Markowitz et al., 1988, J. Virol. 62:1120; U.S. Pat. No. 5,124,263; EP453242, EP178220; Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick,BioTechnology 3 (1985) 689; International Patent Publication No. WO95/07358, published Mar. 16, 1995, by Webster, K. A., Kubasiak, L. A.,Prentice, H. and Bishopric, N. H.: Stable germline transmission of ahypoxia-activated molecular gene switch. From the double helix tomolecular medicine, (ed. W. J. Whelan et al.), Oxford University Press,(2003); and Kuo et al., 1993, Blood 82:845.

The retroviruses are integrating viruses that infect dividing cells. Theretrovirus genome includes two LTRs, an encapsidation sequence and threecoding regions (gag, pol and env). In recombinant retroviral vectors,the gag, pol and env genes are generally deleted, in whole or in part,and replaced with a heterologous nucleic acid sequence of interest.These vectors can be constructed from different types of retrovirus,such as, HIV, MoMuLV (“murine Moloney leukaemia virus” MSV (“murineMoloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV (“spleennecrosis virus”); RSV (“Rous sarcoma virus”) and Friend virus. Defectiveretroviral vectors are disclosed in WO95/02697.

Retroviral vectors can be constructed to function as infectiousparticles or to undergo a single round of transfection. In the formercase, the virus is modified to retain all of its genes except for thoseresponsible for oncogenic transformation properties, and to express theheterologous gene. Non-infectious viral vectors are prepared to destroythe viral packaging signal, but retain the structural genes required topackage the co-introduced virus engineered to contain the heterologousgene and the packaging signals. Thus, the viral particles that areproduced are not capable of producing additional virus. Targeted genedelivery is described in International Patent Publication WO 95/28494,published October 1995.

Lentiviruses include members of the bovine lentivirus group, equinelentivirus group, feline lentivirus group, ovinecaprine lentivirus groupand primate lentivirus group. The development of lentiviral vectors forgene therapy has been reviewed in Klimatcheva et al., 1999, Frontiers inBioscience 4: 481-496. The design and use of lentiviral vectors suitablefor gene therapy is described, for example, in U.S. Pat. No. 6,207,455,issued Mar. 27, 2001, and U.S. Pat. No. 6,165,782, issued Dec. 26, 2000.Examples of lentiviruses include, but are not limited to, HIV-1, HIV-2,HIV-1/HIV-2 pseudotype, HIV-1/SIV, FIV, caprine arthritis encephalitisvirus (CAEV), equine infectious anemia virus and bovine immunodeficiencyvirus. HIV-1 is preferred.

Host cells can be infected with the subject viral vectors ex vivo or invivo. Suitable host cells infected with the viral vectors includecultured cell lines and cells isolated from living organisms.Preferably, host cells of the subject invention are animal cells, morepreferably mammalian cells. Exemplified mammalian host cells include,but are not limited to, cells derived from apes, chimpanzees,orangutans, humans, monkeys, dogs, cats, horses, cattle, pigs, rabbits,sheep, goats, chickens, mice, rats, guinea pigs, and hamsters.

Alternatively, the vector can be introduced in vivo as nucleic acid freeof transfecting excipients, or with transfection facilitating agents,e.g., lipofection. The use of cationic lipids may promote encapsulationof negatively charged nucleic acids, and also promote fusion withnegatively charged cell membranes [Feigner and Ringold, Science337:387-388 (1989)].

Particularly useful lipid compounds and compositions for transfer ofnucleic acids are described in International Patent PublicationsWO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127. The use oflipofection to introduce exogenous genes into the specific organs invivo has certain practical advantages. Targeted peptides, e.g., hormonesor neurotransmitters, and proteins such as antibodies, or non-peptidemolecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (see InternationalPatent Publication WO95/21931), peptides derived from DNA bindingproteins (see International Patent Publication WO96/25508), or acationic polymer (see International Patent Publication WO95/21931).

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter [see, e.g., Wu et al., J. Biol. Chem. 267:963-967(1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Williams etal., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991)]. Receptor-mediatedDNA delivery approaches can also be used [Curiel et al., Hum. Gene Ther.3:147-154 (1992); Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)].

Formulations and Administration

The expression vectors and therapeutic composition comprising theexpression vectors is administered in an effective amount i.e. an amountsufficient to evoke the desired pharmacological response. This isgenerally an amount sufficient to produce lessening of one or more ofthe effects of pathological vascular proliferation. In the case ofretinopathy, it is an amount sufficient to produce regression and/orinhibition of neovascularization and/or an amount sufficient to produceimproved visual acuity.

In preferred embodiments, the vectors and therapeutic compositions ofthe subject invention are administered to a subject by injection intothe targeted region of the eye. The amount of vector to be delivereddepends on the size of the eye, how extensive an area was being targetedand is readily determined by a practioner. When administered it ispreferred that the vectors be given in a pharmaceutical vehicle suitablefor injection such as a sterile aqueous solution or dispersion.Following administration, the subject is monitored to detect changes ingene expression. Dose and duration of treatment can be determinedindividually depending on the condition or disease to be treated. A widevariety of conditions or diseases can be treated based on the geneexpression produced by administration of the gene of interest in thevector of the subject invention. The dosage of vector delivered usingthe method of the invention may vary, depending on the desired responseby the host and the vector used. Generally, it is expected that up to100-200 μg of DNA or RNA can be administered in a single dosage,although a range of 0.5 mg/kg body weight to 50 mg/kg body weight may besuitable for most applications.

In one embodiment, the subject invention utilizes injection techniquesrequire puncturing layers of the eye, including the sclera, choroid,retina, subretina, etc. In one embodiment, to minimize trauma to thoselayers of the eye, the vectors and therapeutic compositions of thesubject invention can be administered into the sub-tenon (i.e.,episcleral) space surrounding the scleral portion of the eye. Thevectors and therapeutic compositions can also be administered to otherregions of the ocular apparatus such as, for instance, the ocularmuscles, the orbital fascia, the eye lid, the lacrimal apparatus, andthe like as is appropriate.

Preferably, the therapeutic factor or nucleic acid sequence encoding thetherapeutic factor is administered via an ophthalmologic instrument fordelivery to a specific region of an eye. The use of a specializedophthalmologic instrument ensures precise administration of thetherapeutic factor or the nucleic acid sequence encoding the therapeuticfactor, while minimizing damage to adjacent ocular tissue. Delivery ofthe therapeutic factor or nucleic acid sequence encoding the therapeuticfactor to a specific region of the eye also limits exposure ofunaffected cells to the therapeutic factor, thereby reducing the risk ofside effects. A preferred ophthalmologic instrument is a combination offorceps and subretinal needle or sharp bent cannula.

In certain embodiments, the vectors and compositions of the subjectinvention are locally injected into the targeted region of the eye, viaan appropriate route such as subretinal, transscleral, or transcornealadministration.

The vectors and therapeutic compositions provided by the subjectinvention are typically administered to a mammal, particularly a human,dog or cat, any of which is intended to be encompassed by the term“patient” herein, in need of the prevention or treatment of pathologicalvascular proliferation. Pathological conditions involving vascularproliferation include, for example, tumor growth, age-related maculardegeneration (AMD), diabetic retinopathy and retinopathy of prematurity(ROP).

In one method, the subject invention involves identifying an individualwho has, or who is at risk for developing pathological vascularizationand then providing that individual with a composition of the subjectinvention.

The terms “pharmaceutically acceptable carrier” or a “carrier” refer toany generally acceptable excipient or drug delivery device that isrelatively inert and non-toxic. The agent can be administered with orwithout a carrier. When treating retinopathies, a preferred embodimentis to administer the agent to the retinal area or the vasculature aroundor leading to the retina. Suitable carriers (e.g., pharmaceuticalcarriers) include, but are not limited to sterile water, salt solutions(such as Ringer's solution), alcohols, polyethylene glycols, gelatin,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrolidone, etc. Such preparations canbe sterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, coloring, and/oraromatic substances and the like which do not deleteriously react withthe active compounds. They can also be combined where desired with otheractive substances, e.g., enzyme inhibitors, to reduce metabolicdegradation. A carrier (e.g., a pharmaceutically acceptable carrier) ispreferred, but not necessary to administer the agent.

Suitable non-toxic pharmaceutically acceptable carriers for use with theagent will be apparent to those skilled in the art of pharmaceuticalformulation. See, for example, Remington's Pharmaceutical Sciences,seventeenth edition, ed. Alfonso R. Gennaro, Mack Publishing Company,Easton, Pa. (1985).

The therapeutic dosage range can be determined by one skilled in the arthaving the benefit of the current disclosure. Naturally, suchtherapeutic dosage ranges will vary with the size, species and physicalcondition of the patient, the severity of the patient's medicalcondition, the particular dosage form employed, the route ofadministration and the like.

According to the subject invention, the local ocular administration ofactive agent of the invention, and/or formulations thereof, attenuateocular pathological disease processes. Thus, local ocular administrationof the active agent of the invention, and/or formulations thereof,provides for an efficacious but safe controlled administration directlyin the eye.

Ocular therapies, as described herein, provide significant advantagesfor treating neovascular ocular disease relative to current lasersurgery treatment modalities including panretinal photocoagulation,which can be accompanied by extensive ocular tissue damage. In theexamples of posterior neovascular ocular diseases, such as age relatedmacular degeneration and diabetic retinopathy, target ocular pathologiesand tissues for treatment are especially localized to the retinal,choroidal and corneal ocular compartments.

Preferably, the agent is administered locally to the eye, retinal area,choroid area or associated vasculature. The vectors and therapeuticcomposition can be administered in a single dose or in more than onedose over a period of time to confer the desired effect.

In a preferred embodiment, the agents of the subject invention can beformulated for parenteral administration. The preparation of an aqueouscomposition that contains one or more agents, such as a geneticconstruct of the subject invention, will be known to those of skill inthe art in light of the present disclosure. Typically, such compositionscan be prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

Solutions of the active compounds as freebase or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Compositions comprising the agents of the subject invention can beformulated into a composition in a neutral or salt form.Pharmaceutically acceptable salts include the acid addition salts andthose formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts can also be derived from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine, procaine and the like.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingtechniques, which yield a powder of the active ingredient, plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

A further embodiment of the subject invention provides for theadministration of agent compounds in combination with otherpharmacological therapies. Combination therapies with other medicamentstargeting similar or distinct disease mechanisms have advantages ofgreater efficacy and safety relative to respective monotherapies witheither specific medicament.

In one embodiment, the composition is used to treat neovascular oculardisease by localized (for example, in ocular tissue) concurrentadministration with other medicaments that act to block angiogenesis.Medicaments that can be concurrently administered with a peptidecompound of the invention include, but are not limited to, vascularendothelial growth factor VEGF blockers (e.g. by VEGF neutralizingbinding molecules such as MACUGEN® (Eyetech) and LUCENTIS® (ranibizumab,Genentech), Squalamine lactate (Genaera Corporation); and VEGF tyrosinekinase inhibition) for treating neovascular ocular disease (AMD andDiabetic Retinopathy) and glucocorticoids (e.g. Triamcinolone) fortreating macular edema.

When administering more than one, the administration of the agents canoccur simultaneously or sequentially in time. The agents can beadministered before and after one another, or at the same time. Themethods also include co-administration with other drugs that are used totreat retinopathy or other diseases described herein.

Materials and Methods Cell Culture & Hypoxia Treatment

ARPE-19 (human RPE) cells were purchased from American Type CultureCollection (Manassas, Va.). Cell Cultures were propagated and maintainedin Dulbecco's modified eagle medium (DMEM) or DMEM/F-12(Cellgro-Mediatech, Herndon, Va.) and supplemented with 10% fetal bovineserum (HyClone, Logan, Utah) and 1% penicillin/streptomycin(Cellgro-Mediatech). All hypoxia experiments were performed using aShel-Lab Bactron anaerobic glove-box chamber (Sheldon Manufacturing Inc,Cornelius, Oreg.).

Construction of Hypoxia Inducible Promoter (HRSE-6×HRE-RPE65)

The HRSE-6×-RPE65 promoter assembly has been described in Doughent etal. (2008). Briefly, the hypoxia responsive element (HRE) sequence isderived from the human phosphoglycerate kinase (PGK) gene in the senseorientation, 5′-TGT CAC GTC CTG CAC GAC GTA-3′ (SEQ ID NO: 2). Theneuron restrictive silencer element (NRSE) sequence is derived from thehuman synapsin I gene in the sense orientation, 5′-TTC AGC ACC GCG GACAGT GCC-3′(SEQ ID NO: 3) (Brene et al. (2007)). The hypoxia regulatedsilencing element (HRSE) sequence was made by placing three copies ofthe NRSE sequence and three copies of the HRE in an alternating tandemorder. The 6×HRE sequence was made by synthesizing six tandem copies ofthe HRE sequence into a single, 144 bp, double-stranded oligomer. The“6×HRE” sequence includes a Sac I restriction site at each terminus. TheRPE65 promoter sequence is derived from the 5′ upstream sequence (−325to +52) of the RPE65 gene.

The RPE65, HSRE, and 6×HRE sequences were serially subcloned into avector. Specifically, the native RPE65 promoter sequence was insertedinto the vector at a multiple cloning site (MCS). Then the HRSE sequencewas cloned into the MCS at a position just upstream of the RPE65promoter to provide normoxic silencing. Additionally, the 6×HRE sequencewas cloned into the MCS just downstream of the HRSE sequence. The 6×HREsequence provides conditionally-enhanced expression of the transgeneunder hypoxia, during which the transcription factor HIF 1 heterodimertranslocates into the nucleus and binds HREs thereby upregulatingtransgene expression.

Hypoxia-Regulated, RPE-Specific Viral Vectors

Self-complimentary, hypoxia-regulated, and RPE-specific viral vectorswere constructed to drive the expression of the therapeutictransgene—human endostatin gene. FIG. 1 illustrates the vector design.

Specifically, the human endostatin XVIII gene (along with SV40 poly A)was obtained by amplifying the human endostatin gene from thepBLAST42-hEndo XVIII expression vector (InvivoGen, San Diego, Calif.)using polymerase chain reaction (PCR). The scAAV2-CMV-hEndostatin vector(“scAAV2-CMV-Endo”) was constructed by cloning the human endostatinXVIII gene is into a scAAV2 double stranded, self-complimentaryadeno-associated viral vector. The scAAV2-HRES-6×HRE-RPE65-hEndostatinvector (abbreviated as “scAAV2-Regulated-Endostatin”, “scAAV2-Reg-Endo”,or “Reg-Endo”) was constructed by replacing the CMV promoter sequencewith the HRSE-6×-RPE65 promoter sequence in the scAAV2-CMV-hEndo vector.The scAAV2-Regulated-Endostatin vector as well as the scAAV2-CMV-Endovector also contains a GFP sequence downstream of the human endostatingene.

Proper placement and orientation of the promoter elements and theendostatin gene in both scAAV2-CMV-hEndo and scAAV2-Reg-Endo plasmidswere confirmed by sequence analysis (CRC-DNA Sequencing Facility,University of Chicago, Chicago, Ill.) prior to production of viralstocks by the University of North Carolina's Vector Core facility(Chapel Hill, N.C.).

The scAAV2-HRSE-6×HRE-RPE65-Endostatin vector produced a viral titer of1.5×10¹² virus molecules/ml, while the scAAV2-CMV-Endostatin produced aviral titer of 2.0×10¹² virus molecules/ml.

The viral vectors were used in vitro rtPCR analysis of transgeneexpression and in vivo experiments. The scAAV2-CMV-Endostatin vector wasdiluted to producing a viral titer of 1.5×10¹² virus molecules/ml priorto use.

Reverse Transcription Polymerase Chain Reaction (rtPCR)

ARPE-19 cells were grown to confluence in tissue culture-treatedsix-well plates (Corning Life Sciences, Lowell, Mass.), and thentransduced with 1 uL of scAAV-HRES-6×HRE-RPE65-Endostatin (1.5×10¹²virus molecules/ml). The transduced cells and untransduced cells(negative control) were subjected to 24 hours of hypoxia or normoxia.

After incubation, cells were lysed, and total RNA was extracted andpurified using an RNeasy Plus Mini kit (Qiagen, Valencia, Calif.). RNAconcentration was determined by spectrophotometry. RNA wasreverse-transcribed into cDNA using Superscript III ReverseTranscription Supermix (Invitrogen).

PCR was performed using 50 μl solution containing 10 ng cDNA, PlatinumTaq polymerase (Invitrogen), dNTP, MgCl₂, and primers that target eitherthe vector-derived endostatin sequence or the beta-actin sequence. Theendostain primer pair amplifies a 682 base pair region of thevector-derived endostain gene that starts within the endostatin poly Aregion and ends within the GFP portion of the vector-derived endostaintranscript, and has the following sequences: 5′-GAACAGCTTCATGACTGC-3′(SEQ ID NO:4) (Forward) and 5′-GGTGCAGATGAACTTCAG-3′ (SEQ ID NO:5)(Reverse). The beta-actin primer pair amplifies a 367 base pair regionwithin the beta-actin transcript, and has the following sequences 5′TCTACAATGAGCTGCGTGTG 3′ (SEQ ID NO:6) (Forward) and5′-GGTGCAGATGAACTTCAG-3′ (SEQ ID NO:7) (Reverse). The beta-actintranscripts, which are endogenous and constitutively expressed, serve asthe reaction and loading control.

After an initial incubation of 1 minute at 94° C., 35 cycles of PCRamplification (94° C. for 30 seconds, 49° C. for 30 seconds, 72° C. for1 minute) were performed using a thermocycler (Eppendorf, Hauppauge,N.Y.). 25 μl of each amplified product was loaded into a 1% agarose gel(containing ethidium bromide) and resolved by electrophoresis. Bandswere imaged using the Gel-Doc-it system and LabWorks software (UVP,Upland, Calif.).

Animals

Female C57BL/J6 mice were used in the study. Because age is acontributing factor in AMD, all mice used for the AMD study were 7-9months old (retired breeders). Mice were handled using proceduresconsistent with the ARVO Statement for the Use of Animals in Ophthalmicand Vision Research and approved by the FAU and University of MiamiInstitutional Animal Care and Use Committees (IACUC).

Subretinal Injections

Mouse pupils were dilated with 1% tropicamide, and the mice weredark-adapted prior to anesthetization. Anesthetization was performed byintraperitoneal injection of 80-100 mg/kg ketamine and 10 mg/kg ofxylazine. After anesthetization, proparacaine hydrochloride localanesthesia was applied to each eye.

A pilot hole was made on the nasal cornea, just inside the pupil, with a30G1/2 disposable needle. A 33-gauge, blunt-ended needle mounted on a 10ul Hamilton syringe was introduced through the corneal opening,carefully avoiding the lens, stopping at the anterior retina. Onemicroliter of scAAV2-HRSE-6×HRE-RPE65-Endostatin, scAAV2-CMV-Endostatin(both 1.5×10¹² virus molecules/ml), or vehicle (PBS) was slowly injectedinto the subretinal space. One eye was injected with eitherscAAV2-CMV-GFP or scAAV2-HRES-6×HRE-RPE65-Endostatin, and thecontralateral eye was injected with vehicle. Successful injections wereconfirmed upon fundus visualization of sub-retinal blebs, indicatingretinal detachment. Such detachments are typically transient and areresolved within 24 hrs. Following injections, 1% atropine eye drops andneomycin/polymixin B/dexamethasone ophthalmic ointments wereadministered.

Mouse Model of Choroidal Neovascularization

The laser-induced murine model of CNV has been previously described inKasman et al. (2008). One week following subretinal injection, pupilswere dilated with 1% tropicamide, and mice were anesthetized with 80-100mg/kg ketamine-10 mg/kg of xylazine by intraperitoneal injection. CNVwas induced by irradiating mouse eye tissues with an argon 532 nm greendiode laser (100 uM spot size, 150 mW intensity, 0.1 s duration—NidekGYC-1000, Fremont, Calif.) mounted on a Haag-Streit slit lamp, using acoverslip as a contact lens.

Three lesions were made at 3, 6, and 9 o'clock positions between retinalvessels and 2-3 disc diameters away from the optic nerve. Formation of abubble at the time of laser application indicated rupture of Bruch'smembrane, which is an important factor for CNV induction. Only lesionsthat produce bubble(s) were assessed in this invention.

Histological Evaluation of Choroidal Neovascularization: RPE Flatmounts

Fourteen days post-laser treatment, mice were anesthetized with 80-100mg/kg of ketamine and 10 mg/kg of xylazine. Vasculature was stained with100 uL of 1 mg/mL fluorescein- or DyLight 594-conjugated L. esculentum(tomato) lectin (Vector Laboratories, Burlingame Calif.) in PBS viacardiac perfusion. Five minutes later, the mice were sacrificed by 100%CO₂ and the eyes were enucleated and placed in 1×PBS. A hole was made inthe cornea prior to fixing in 4% PFA for one hour. The anterior chamber,lens and cornea were dissected out, and the neurosensory retina wascarefully removed from the RPE layer. The remaining RPE, choroid, scleracomplex was flatmounted on a slide by making 3-4 radial incisions, beingcareful to avoid any lesions. The retinal flatmounts were coverslippedusing Vectashield hard-set mounting media with DAPI counterstain (VectorLaboratories, Burlingame, Calif.).

Retinal flatmounts were observed using a Leica SP5 confocal microscope(Leica Microsystems, Wetzlar, Germany) with a 20× objective under FITC(excitation at 488 nm and emission at 535 nm) and DAPI (excitation at350 nm and emission at 470 nm) filters. Compressed 20 um Z-stack images(1 um/frame) were obtained for each lesion using Leica's LAS software(Leica Microsystems). CNV area for each lesion was quantified usingImage J (National Institute of Health, Bethesda, Md.).

The initial number of CNV lesions associated with bubble formation uponlaser application was 331 (138 vehicle treated, 169 Reg-Endo treated,and 24 CMV-Endo treatment). The following exclusion and inclusioncriteria for CNV lesions and area measurements were developed andimplemented by an investigator masked with respect to treatment groups.CNV lesions associated with retinal bleeding from the site of laserapplication were excluded; this is indicative of the laser rupturing aminor retinal vessel (since major vessels are easily seen and avoided)and typically caused fusion of the ruptured retinal vessel(s) with theunderlying CNV growth. Additionally, lesions that had variousdiscrepancies with flatmount preparation (i.e. adherence to neurosensoryretina) and complications that interfered with microscopic documentation(i.e. weak/blurred lectin staining and inadequate imaging) wereexcluded.

The remaining lesions were included into the raw data set, which wascomposed of 58 vehicle treated, 78 Reg-Endo treated, and 19 CMV-Endotreated lesions stored as 20× compressed Z-stack confocal micrographs.After 3 independent measurements of CNV area were made, for each lesionin the raw data set, the final exclusions were made to create the finaldata set, which was used to calculate the statistical significancebetween treatment groups. The final data set excluded coalesced lesionsthat had no discernable boundaries and lesions that contained vessels orportions of vessels from the neurosensory retina. Also, outliers wereidentified, and eliminated if the percentage of standard deviation forthe 3 area measurements was greater than 15% of their average.

The final data set included 53 vehicle treated, 53 Reg-Endo treated, and17 CMV-Endo treated lesions. The average of the 3 independent CNV areameasurements for each lesion was used for data analysis.

Statistical analysis was made using a student's paired t test(2-tailed), for either AAV2-CMV-Endostatin orAAV2-HRSE-HRE-RPE65-Endostatin versus vehicle control. Mean CNV areaswere also compared between the eyes that were injected withAAV2-HRES-6×HRE-RPE65-Endostatin and the vehicle injected eyes that hadtwo or more quantifiable lesions for each eye.

Histological Evaluation of Choroidal Neovascularization: Cryosections

Mice were subretinally injected with AAV2-HRSE-6×HRE-RPE65-Endostatin,scAAV2-CMV-Endostatin, or vehicle. CNV was induced 1 week followingsubretinal injection. Eyes were harvested from mice at 3 days post-laserinjury. Enucleated eyes were fixed in 4% paraformaldehyde in PBS for 1hr, cryoprotected overnight in 30% sucrose in PBS, embedded inTissue-Tek optimal cutting temperature (OCT), snap frozen in liquidnitrogen, and stored at −80° C.

8 μm serial cryosections were mounted on charged slides and brieflywashed in PBS. Tissues were then blocked for 2 hrs with PBS containing5% horse serum and 0.1% Triton X-100. Sections were incubated in primaryantibody (goat anti-human endostatin; 1:100 in blocking buffer, R&DSystems, Minneapolis, Minn.) overnight at 4° C. Then, the sections werewashed with PBS/0.1% Triton X-100, and incubated in donkeyanti-goat-Alexa-488 secondary antibody (1:1000 in PBS, Molecular Probes,Eugene, Oreg.) for two hours at room temperature. The sections were thenwashed with PBS/0.1% Triton X-100, and the slides were air-dried priorto coverslipping with Vectashield Hard Set Mounting Medium with DAPI(Vector Laboratories, Inc., Burlingame, Calif.).

The sections were imaged with a Nikon Eclipse TE2000-S invertedfluorescent microscope (Nikon Instruments Inc., Melville, N.Y.) usingFITC and DAPI filters. All images were captured at 10× and 20× under thesame conditions using Nikon's NIS Imaging software.

EXAMPLES

Following are examples that illustrate embodiments for practicing theinvention. The examples should not be construed as limiting.

Example 1 Hypoxia-Induced Production of Endostatin Transcripts

This Example shows that the scAAV-HRES-6×HRE-RPE65-Endostatin viralvector produces viable endostatin transcripts in RPE cells under hypoxiabut not under normoxia.

FIG. 1 illustrates the design of viral vectors used in the Examples.ARPE-19 cells were transduced with the scAAV-HRES-6×HRE-RPE65-endostatin(“Reg-Endo”) vector, and the transduced cells were exposed to 24 hoursof hypoxia or normoxia.

RtPCR was performed using primers that selectively amplify a 682 bpregion of cDNA of the Reg-Endo vector. The amplified region beginswithin the endostatin poly adenylation signal and terminates within theGFP sequence. The presence of the GFP sequence, which is not endogenousto ARPE-19 cells, can be detected rtPCR; Cells that produce transcriptscontaining the GFP sequence contain the Reg-Endo vector.

The results show that there is very minimal expression of virus-derivedendostatin mRNA in normoxia, and a high level of endostain expressionfollowing hypoxic exposure (FIG. 2). The PCR products are of the samesize as products amplified from both the purified Reg-Endo plasmid andthe denatured Reg-Endo virus.

In addition, rtPCR was performed using mRNA isolated from untransducedARPE-19 cells. No virus-derived endostatin transcripts were detectedfrom in cells placed under hypoxia (FIG. 2) as well as normoxia.

Example 2 Reduction of Laser-Induced CNV by Hypoxia-Regulated,RPE-Specific Endostatin Gene Therapy

This Example shows that hypoxia-regulated, RPE-specific endostatin genetherapy effectively inhibits chorodial neovascularization (CNV), and canbe used to prevent and treat ocular neovascular diseases such asage-related muscular degeneration (AMD).

Briefly, 1 uL (1.5×10⁹ virus molecules) of the AAV2-Reg-Endo vector, theAAV2-CMV-Endo vector, or vehicle (VC) was subretinally injected into thesubretinal space of C57BL/J6 mice via the trans-corneal route. Nearly100% of RPE transduction was observed three weeks after injection. AscAAV2-CMV-GFP vector was used to confirm transduction efficiency priorto the use of the endostatin vectors.

One week following subretinal injection, mouse eye tissues areirradiated with a laser beam of 532 nm (100 μm/150 mW) to induce CNV bylaser rupture of Bruch's membrane. The laser-induced murine model of CNVis an art-recognized animal model of AMD.

Confocal microscopy of RPE flatmounts was performed two weeks followinglaser injury. The two-week time period provided sufficient time for CNVdevelopment and coincided with the three week period for maximal vectorexpression.

The mean area of laser-induced CNV lesions following subretinalinjection of scAAV2-HRES-6×HRE-RPE65-Endostatin was compared to that ofvehicle-injected, and scAAV2-CMV-Endostatin-injected retinas. The CMVpromoter is not cell-specific and is considered one of the mostconstitutively active promoters used in gene therapy; therefore, thelevel of endostain production in the eyes injected with thescAAV2-CMV-Endostatin vector can indicate whether the hypoxia-regulated,RPE-specific promoter produce sufficient amount of endostatin to reduceCNV lesion area. In addition, the scAAV2-CMV-Endostatin vector, whichcontains the ubiquitously-expressed CMV promoter, would indicate whetherRPE is an appropriate area of the retina for endostatin expression at alevel that is effective for reduction of CNV growth.

FIG. 3 shows representative confocal micrographs of CNV lesions of theeyes injected with the AAV2-Reg-Endo vector, the AAV2-CMV-Endo vector,or vehicle (VC). The lesions in the vehicle-injected eyes were visiblymuch larger, with more tortuous neovascular growth (green “vessels” inFIG. 3) than the endostatin vector-injected group. Subretinaladministration of AAV2-HRES-6×HRE-RPE65-Endostatin resulted in aremarkable, 80% reduction in mean CNV area, compared to lesions fromvehicle injected eyes (51 435±3 535 um² vs. 257 701±15 310 um²; P<0.001;FIG. 4A). Subretinal injection of AAV2-CMV-Endostatin reduced mean CNVarea by 75.8%, compared to lesions from vehicle injected eyes (62 345±9354 um² vs. 257 701±15 310 um²; P<0.001).

This Example also compares mean CNV areas of lesions in the eyesinjected with the scAAV2-HRES-6×HRE-RPE65-Endostatin vector with that ofthe vehicle injected eyes that had at least two measurable lesions pereye (FIGS. 4B & C). This comparison eliminates variability betweenanimals, thereby making a more accurate assessment of lesion areasbetween treatment groups. Mean CNV areas in the Regulated-Endostatintreated eyes were at least 76% smaller than that of the vehicle-injectedcontralateral eyes (56 332±19 991 um² vs. 231 275±84 548 um²; P<0.001).

Each sample of FIG. 5 represents 4 homogenized posterior eyecups pooledfor each of the 3 treatment groups at 3 time points: pre-laser (5 dayspost-injection), 3 days post-laser, and 2 weeks post-laser. Endostatinexpression from the hypoxia regulated vector is initiated by laserinjury to the retina, and is reduced 2 weeks following laser induction(FIG. 5). Specifically, FIG. 5 shows that there is conditional silencingof the Reg-Endo vector before laser injury, then enhanced activationsubsequent to laser injury, and a return to baseline 2 weeks post-laserinjury.

In comparison, for mice injected with the CMV-Endo vector, endostatinconcentrations are elevated prior to laser injury and remain elevatedthrough the two-week post-laser time point. Constitutive endostatinexpression in retinal tissues can produce detrimental effects tonon-pathological tissues. For example, in non-diseased regions of theeye, elevated endostatin could have a range of effects that may alterendogenous cell-to-cell communication and interactions.

The CNV lesion histology light micrographs (FIG. 6) also show that thehypoxia-regulated, RPE-specific endostatin(scAAV2-HRES-6×HRE-RPE65-Endostatin) gene therapy significantly reducedCNV lesion size.

Example 3 Immunohistochemical Localization of Regulated Expression ofEndostatin Protein

Immunohistochemistry of CNV lesions obtained 3 days post-laser injuryconfirmed that expression of human endostatin protein is focallyelevated within the area of laser damage (FIG. 7). Endostatin expressionwas localized at the RPE/choroid interface, which indicates itsexpression and secretion from the basal side RPE subsequent tolaser-induced inflammation and hypoxia. No detectable level ofendostatin was observed in unlasered eyes treated withscAAV2-Regulated-Endostatin.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

REFERENCES

-   Bainbridge, J. W., A. Mistry, et al. (2003). “Hypoxia-regulated    transgene expression in experimental retinal and choroidal    neovascularization.” Gene Ther 10(12): 1049-1054.-   Balaggan, K. S., K. Binley, et al. (2006). “EIAV vector-mediated    delivery of endostatin or angiostatin inhibits angiogenesis and    vascular hyperpermeability in experimental CNV.” Gene Ther 13(15):    1153-1165.-   Cideciyan, A. V., W. W. Hauswirth, et al. (2009). “Human RPE65 gene    therapy for Leber congenital amaurosis: persistence of early visual    improvements and safety at 1 year.” Hum Gene Ther 20(9): 999-1004.-   Dougherty, C. J., Smith, G. W., Prentice, H. M., Dorey, C. K.,    Webster, K. A. and Blanks, J. C. Robust Hypoxia-Selective Regulation    of an RPE-Specific AAV Vector. Molecular Vision (2008).-   Kachi, S., K. Binley, et al. (2009). “Equine infectious anemia viral    vector-mediated codelivery of endostatin and angiostatin driven by    retinal pigmented epithelium-specific VMD2 promoter inhibits    choroidal neovascularization.” Hum Gene Ther 20(1): 31-39.-   Mori, K., A. Ando, et al. (2001) “Inhibition of choroidal    neovascularization by intravenous injection of adenoviral vectors    expressing secretable endostatin.” Am J Pathol 159(1): 313-320.-   Ruan, H., H. Su, et al. (2001). “A hypoxia-regulated    adeno-associated virus vector for cancer-specific gene therapy.”    Neoplasia 3(3): 255-263.-   Tang, Y. L., Y. Tang, et al. (2005). “A hypoxia-inducible vigilant    vector system for activating therapeutic genes in ischemia.” Gene    Ther 12(15): 1163-1170.-   Boulanger A, Liu S, Henningsgaard A A, Yu S, Redmond T M. The    upstream region of the Rpe65 gene confers retinal pigment    epithelium-specific expression in vivo and in vitro and contains    critical octamer and E-box binding sites. J Biol Chem 2000; 275(40):    31274-82.-   Brene S, Messer C, Okado H, Hartley M, Heinemann S F, Nestler E J.    Regulation of GluR2 promoter activity by neurotrophic factors via a    neuron-restrictive silencer element. Eur J Neurosci 2000; 12(5):    1525-33.-   Kong F, Li W, Li X, Zheng Q, Dai X, Zhou X et al. Self-complementary    AAV5 vector facilitates quicker transgene expression in    photoreceptor and retinal pigment epithelial cells of normal mouse.    Exp Eye Res 2010; 90(5): 546-54.

We claim:
 1. A method of reducing or preventing ocularneovascularization in a subject, wherein the method comprisesadministering, into a target ocular tissue of the subject, an expressionvector comprising a transgene encoding a therapeutic molecule, whereinthe transgene is operably linked to an ocular-specific promoter and isplaced under the control of a hypoxia-responsive element, wherein theocular-specific promoter selectively drives gene expression in theocular tissue having, or at risk of developing, neovascularization,wherein the hypoxia-responsive element upregulates gene expression underhypoxia but not under nomoxia, whereby ocular neovascularization isreduced or prevented.
 2. The method of claim 1, wherein thehypoxia-responsive element comprises a hypoxia response element (HRE)sequence.
 3. The method of claim 2, wherein the hypoxia-responsiveelement comprises at least three copies of the hypoxia response element(HRE) sequence.
 4. The method of claim 2, wherein the HRE sequencecomprises SEQ ID NO:1 or SEQ ID NO:2.
 5. The method of claim 1, whereinthe expression vector further comprises an aerobic silencer thatsilences gene expression under normoxia.
 6. The method of claim 5,wherein the aerobic silencer comprises a neuron-restrictive silencerelement (NRSE) sequence.
 7. The method of claim 5, wherein the aerobicsilencer comprises at least two copies of a hypoxia response element(HRE) sequence and at least two copies of a neuron-restrictive silencerelement (NRSE) sequence, wherein the HRE and the NRSE are placed inalternating tandem order.
 8. The method of claim 1, wherein theocular-specific promoter selectively drives gene expression in retinalcells, retinal epithelial cells, Muller cells, or choroid cells.
 9. Themethod of claim 8, wherein the ocular-specific promoter selectivelydrives gene expression in retinal epithelial cells.
 10. The method ofclaim 1, wherein the ocular-specific promoter is a RPE65 promotersequence.
 11. The method of claim 5, wherein the expression vectorcomprises, in the 5′ to 3′ direction, an aerobic silencer comprising atleast two copies of a hypoxia response element (HRE) sequence and atleast two copies of a neuron-restrictive silencer element (NRSE)sequence, wherein the HRE and the NRSE are placed in alternating tandemorder; at least three copies of the hypoxia response element (HRE)sequence; a RPE65 promoter sequence; and a transgene gene encodingendostatin.
 12. The method of claim 1, wherein the therapeutic moleculeis an angiogenesis inhibitor.
 13. The method of claim 12, wherein theangiogenesis inhibitor is selected from endostatin, a fibroblast growthfactor (FGF) or VEGF receptor, angiostatin, pigment epithelium-derivedfactor (PEDF), and platelet factor 4 (PF-4).
 14. The method of claim 13,wherein the angiogenesis inhibitor is endostatin.
 15. The method ofclaim 1, wherein the expression vector is an AAV vector.
 16. The methodof claim 1, used to prevent or treat choroidal neovascularization. 17.The method of claim 1, used to prevent or treat age-related maculardegeneration, histoplasmosis, myopic degeneration, choroidal rupture,photocoagulation, choroidal hemangioma, choroidal nonperfusion,choroidal osteomas, choroideremia, retinal detachment,neovascularization at ora serrata, punctate inner choroidopathy,radiation retinopathy, and/or retinal cryoinjury.
 18. The method ofclaim 1, used to prevent or treat retinal and/or cornealneovascularization.
 19. The method of claim 1, wherein the expressionvector is administered via subretinal injection.
 20. An AAV vectorcomprising, in the 5′ to 3′ direction, an aerobic silencer comprising atleast two copies of a hypoxia response element (HRE) sequence and atleast two copies of a neuron-restrictive silencer element (NRSE)sequence, wherein the HRE sequence and the NRSE sequence are placed inalternating tandem order; at least three copies of the hypoxia responseelement (HRE) sequence; a RPE65 promoter sequence; and a transgene geneencoding endostatin; wherein the endostatin transgene is operably linkedto the RPE65 promoter sequence and is placed under the control of theaerobic silencer and the HRE sequence.